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Biophysical Journal Jun 2021Cytosine methylated at the five-carbon position is the most widely studied reversible DNA modification. Prior findings indicate that methylation can alter mechanical...
Cytosine methylated at the five-carbon position is the most widely studied reversible DNA modification. Prior findings indicate that methylation can alter mechanical properties. However, those findings were qualitative and sometimes contradictory, leaving many aspects unclear. By applying single-molecule magnetic force spectroscopy techniques allowing for direct manipulation and dynamic observation of DNA mechanics and mechanically driven strand separation, we investigated how CpG and non-CpG cytosine methylation affects DNA micromechanical properties. We quantitatively characterized DNA stiffness using persistence length measurements from force-extension curves in the nanoscale length regime and demonstrated that cytosine methylation results in longer contour length and increased DNA flexibility (i.e., decreased persistence length). In addition, we observed the preferential formation of plectonemes over unwound single-stranded "bubbles" of DNA under physiologically relevant stretching forces and supercoiling densities. The flexibility and high structural stability of methylated DNA is likely to have significant consequences on the recruitment of proteins recognizing cytosine methylation and DNA packaging.
Topics: Cytosine; DNA; DNA Methylation; Micromanipulation; Nanotechnology
PubMed: 33838135
DOI: 10.1016/j.bpj.2021.03.039 -
Current Opinion in Chemical Biology Aug 2013A subset of enzymes that belong to the radical S-adenosylmethionine (SAM) superfamily is able to catalyze methylation reactions. Substrates of these enzymes are distinct... (Review)
Review
A subset of enzymes that belong to the radical S-adenosylmethionine (SAM) superfamily is able to catalyze methylation reactions. Substrates of these enzymes are distinct from the nucleophilic substrates that undergo methylation by a polar mechanism. Recently, activities of several radical SAM methylating enzymes have been reconstituted in vitro and their mechanisms of catalysis investigated. The RNA modifying enzymes RlmN and Cfr catalyze methylation via a methyl synthase mechanism. These enzymes use SAM in two distinct roles: as a source of a methyl group transferred to a conserved cysteine and as a source of 5'-deoxyadenosyl radical (5'-dA). Hydrogen atom abstraction by this species generates a thiomethylene radical which adds into the RNA substrate, forming an enzyme-substrate covalent adduct. In another recent study, methylation of the indole moiety of tryptophan by the radical SAM and cobalamin-binding domain enzyme TsrM has been reconstituted. Methylcobalamin serves as an intermediate methyl donor in TsrM, and is proposed to transfer the methyl group as a methyl radical. Interestingly, despite the presence of the radical SAM motif, no reductive cleavage of SAM has been observed in this methylation. These important reconstitutions set the stage for further studies on mechanisms of radical methylation.
Topics: Free Radicals; Methylation; RNA; S-Adenosylmethionine; Substrate Specificity; Tryptophan
PubMed: 23835516
DOI: 10.1016/j.cbpa.2013.05.032 -
GigaScience 2015DNA methylation has important roles in the regulation of gene expression and cellular specification. Reduced representation bisulfite sequencing (RRBS) has prevailed in... (Review)
Review
BACKGROUND
DNA methylation has important roles in the regulation of gene expression and cellular specification. Reduced representation bisulfite sequencing (RRBS) has prevailed in methylation studies due to its cost-effectiveness and single-base resolution. The rapid accumulation of RRBS data demands well designed analytical tools.
FINDINGS
To streamline the data processing of DNA methylation from multiple RRBS samples, we present a flexible pipeline named SMAP, whose features include: (i) handling of single-and/or paired-end diverse bisulfite sequencing data with reduced false-positive rates in differentially methylated regions; (ii) detection of allele-specific methylation events with improved algorithms; (iii) a built-in pipeline for detection of novel single nucleotide polymorphisms (SNPs); (iv) support of multiple user-defined restriction enzymes; (v) conduction of all methylation analyses in a single-step operation when well configured.
CONCLUSIONS
Simulation and experimental data validated the high accuracy of SMAP for SNP detection and methylation identification. Most analyses required in methylation studies (such as estimation of methylation levels, differentially methylated cytosine groups, and allele-specific methylation regions) can be executed readily with SMAP. All raw data from diverse samples could be processed in parallel and 'packetized' streams. A simple user guide to the methylation applications is also provided.
Topics: Algorithms; DNA Methylation; Polymorphism, Single Nucleotide; Sulfites
PubMed: 26140213
DOI: 10.1186/s13742-015-0070-9 -
Epigenetics Dec 2019Increasing numbers of studies implicate abnormal DNA methylation in cancer and many non-malignant diseases. This is consistent with numerous findings about... (Review)
Review
Increasing numbers of studies implicate abnormal DNA methylation in cancer and many non-malignant diseases. This is consistent with numerous findings about differentiation-associated changes in DNA methylation at promoters, enhancers, gene bodies, and sites that control higher-order chromatin structure. Abnormal increases or decreases in DNA methylation contribute to or are markers for cancer formation and tumour progression. Aberrant DNA methylation is also associated with neurological diseases, immunological diseases, atherosclerosis, and osteoporosis. In this review, I discuss DNA hypermethylation in disease and its interrelationships with normal development as well as proposed mechanisms for the origin of and pathogenic consequences of disease-associated hypermethylation. Disease-linked DNA hypermethylation can help drive oncogenesis partly by its effects on cancer stem cells and by the CpG island methylator phenotype (CIMP); atherosclerosis by disease-related cell transdifferentiation; autoimmune and neurological diseases through abnormal perturbations of cell memory; and diverse age-associated diseases by age-related accumulation of epigenetic alterations.
Topics: DNA Methylation; Epigenesis, Genetic; Humans; Immune System Diseases; Neoplasms; Nervous System Diseases
PubMed: 31284823
DOI: 10.1080/15592294.2019.1638701 -
Journal of Molecular Biology Oct 2014Protein methylation plays an integral role in cellular signaling, most notably by modulating proteins bound at chromatin and increasingly through regulation of... (Review)
Review
Protein methylation plays an integral role in cellular signaling, most notably by modulating proteins bound at chromatin and increasingly through regulation of non-histone proteins. One central challenge in understanding how methylation acts in signaling is identifying and measuring protein methylation. This includes locus-specific modification of histones, on individual non-histone proteins, and globally across the proteome. Protein methylation has been studied traditionally using candidate approaches such as methylation-specific antibodies, mapping of post-translational modifications by mass spectrometry, and radioactive labeling to characterize methylation on target proteins. Recent developments have provided new approaches to identify methylated proteins, measure methylation levels, identify substrates of methyltransferase enzymes, and match methylated proteins to methyl-specific reader domains. Methyl-binding protein domains and improved antibodies with broad specificity for methylated proteins are being used to characterize the "protein methylome". They also have the potential to be used in high-throughput assays for inhibitor screens and drug development. These tools are often coupled to improvements in mass spectrometry to quickly identify methylated residues, as well as to protein microarrays, where they can be used to screen for methylated proteins. Finally, new chemical biology strategies are being used to probe the function of methyltransferases, demethylases, and methyl-binding "reader" domains. These tools create a "system-level" understanding of protein methylation and integrate protein methylation into broader signaling processes.
Topics: Animals; Chromosomal Proteins, Non-Histone; Electrophoresis, Polyacrylamide Gel; Histones; Humans; Immunoprecipitation; Methylation; Protein Processing, Post-Translational; Proteome; Proteomics
PubMed: 24805349
DOI: 10.1016/j.jmb.2014.04.024 -
PloS One 2022Testing for disease-related DNA methylation changes provides clinically relevant information in personalized patient care. Methylation-Sensitive High-Resolution Melting...
Testing for disease-related DNA methylation changes provides clinically relevant information in personalized patient care. Methylation-Sensitive High-Resolution Melting (MS-HRM) is a method used for measuring methylation changes and has already been used in diagnostic settings. This method utilizes one set of primers that initiate the amplification of both methylated and non-methylated templates. Therefore, the quantification of the methylation levels using MS-HRM is hampered by the PCR bias phenomenon. Some approaches have been proposed to calculate the methylation level of samples using the high-resolution melting (HRM) curves. However, limitations of the methylation calculation using MS-HRM have not been evaluated systematically and comprehensively. We used the Area Under the Curve (AUC), a derivative of the HRM curves, and least square approximation (LSA) to establish a procedure that allowed us to infer methylation levels in an MS-HRM experiment and assess the limitations of that procedure for the assays' specific methylation level measurement. The developed procedure allowed, with certain limitations, estimation of the methylation levels using HRM curves.
Topics: DNA Methylation; DNA Primers; Humans; Polymerase Chain Reaction
PubMed: 36067175
DOI: 10.1371/journal.pone.0273058 -
Epigenetics Dec 2023Abnormal DNA methylation is a fundamental characterization of epigenetics in cancer. Here we demonstrate that aberrant DNA methylating can modulate the tumour immune...
Abnormal DNA methylation is a fundamental characterization of epigenetics in cancer. Here we demonstrate that aberrant DNA methylating can modulate the tumour immune microenvironment in 16 cancer types. Differential DNA methylation in promoter region can regulate the transcriptomic pattern of immune-related genes and DNA hypomethylation mainly participated in the processes of immunity, carcinogenesis and immune infiltration. Moreover, many cancer types shared immune-related functions, like activation of innate immune response, interferon gamma response and NOD-like receptor signalling pathway. DNA methylation can further help identify molecular subtypes of kidney renal clear cell carcinoma. These subtypes are characterized by DNA methylation pattern, major histocompatibility complex, cytolytic activity and cytotoxic t lymphocyte and tumour mutation burden, and subtype with hypomethylation pattern shows unstable immune status. Then, we investigate the DNA methylation pattern of exhaustion-related marker genes and further demonstrate the role of hypomethylation in tumour immune microenvironment. In summary, our findings support the use of hypomethylation as a biomarker to understand the mechanism of tumour immune environment.
Topics: Humans; DNA Methylation; Neoplasms; Epigenesis, Genetic; Biomarkers, Tumor; Immunity; Tumor Microenvironment
PubMed: 36945884
DOI: 10.1080/15592294.2023.2192894 -
Clinical Epigenetics Nov 2022The factors affecting cardioprotective collateral circulation are still incompletely understood. Recently, characteristics, such as CpG methylation of cell-free DNA...
BACKGROUND
The factors affecting cardioprotective collateral circulation are still incompletely understood. Recently, characteristics, such as CpG methylation of cell-free DNA (cfDNA), have been reported as markers with clinical utility. The aim of this study was to evaluate whether cfDNA methylation patterns are associated with the grade of coronary collateral circulation (CCC).
RESULT
In this case-control study, clinical and angiographic data were obtained from 143 patients (mean age, 58 years, male 71%) with chronic total coronary occlusion. Enzymatic methyl-sequencing (EM-seq) libraries were prepared using the cfDNA extracted from the plasma. Data were processed to obtain the average methylation fraction (AMF) tables of genomic regions from which blacklisted regions were removed. Unsupervised analysis of the obtained AMF values showed that some of the changes in methylation were due to CCC. Through random forest preparation process, 256 differentially methylated region (DMR) candidates showing strong association with CCC were selected. A random forest classifier was then constructed, and the area under the curve of the receiver operating characteristic curve indicated an appropriate predictive function for CCC. Finally, 20 DMRs were identified to have significantly different AMF values between the good and poor CCC groups. Particularly, the good CCC group exhibited hypomethylated DMRs. Pathway analysis revealed five pathways, including TGF-beta signaling, to be associated with good CCC.
CONCLUSION
These data have demonstrated that differential hypomethylation was identified in dozens of cfDNA regions in patients with good CCC. Our results support the clinical utility of noninvasively obtained epigenetic signatures for predicting collateral circulation in patients with vascular diseases.
Topics: Humans; Male; Middle Aged; Case-Control Studies; Cell-Free Nucleic Acids; Collateral Circulation; Coronary Artery Disease; DNA Methylation; Female
PubMed: 36320085
DOI: 10.1186/s13148-022-01349-w -
Current Opinion in Chemical Biology Dec 2017RNA methylation is an abundant modification identified in various RNA species in both prokaryotic and eukaryotic organisms. However, the functional roles for the... (Review)
Review
RNA methylation is an abundant modification identified in various RNA species in both prokaryotic and eukaryotic organisms. However, the functional roles for the majority of these methylations remain largely unclear. In eukaryotes, many RNA methylations have been suggested to participate in fundamental cellular processes. Mutations in eukaryotic RNA methylating enzymes, and a consequent change in methylation, can lead to the development of diseases and disorders. In contrast, loss of RNA methylation in prokaryotes can be beneficial to microorganisms, especially under antibiotic pressure. Here we discuss several recent advances in understanding mutational landscape of both eukaryotic and prokaryotic RNA methylating enzymes and their relevance to disease and antibiotic resistance.
Topics: Animals; Disease; Enzymes; Humans; Methylation; Mutation; RNA; Ribosomes
PubMed: 29059606
DOI: 10.1016/j.cbpa.2017.10.002 -
International Journal of Environmental... Dec 2019Arsenic (As) poses a risk to the human health in excess exposure and microbes play an important role in the toxicity of As. Arsenic methylation mediated by microbes is a... (Review)
Review
Arsenic (As) poses a risk to the human health in excess exposure and microbes play an important role in the toxicity of As. Arsenic methylation mediated by microbes is a key driver of As toxicity in the environment and this paper reviews the role of microbial arsenic methylation and volatilization in the biogeochemical cycle of arsenic. In specific, little is presently known about the molecular mechanism and gene characterization of arsenic methylation. The uptake of methylated arsenic in plants is influenced by microbial arsenic methylation in soil, thus enhancing the volatilization of methylated arsenic is a potential mitigation point for arsenic mobility and toxicity in the environment. On the other hand, the potential risk of methylated arsenic on organisms is also discussed. And the directions for future research, theoretical reference for the control and remediation of arsenic methylation, are presented.
Topics: Arsenic; Biological Transport; Methylation; Plants; Soil Microbiology; Soil Pollutants
PubMed: 31835448
DOI: 10.3390/ijerph16245012